Synthesis of biologically active peptides

ABSTRACT

A new peptide which has calcitonin-like biological activity which has a shorter amino acid chain and which is chemically more stable than natural calcitonins. Also resin peptides are disclosed which may be converted to peptides having biological activity and processes for producing said resin peptides and said peptides having biological activity.

This is a division of application Ser. No. 927,456, filed July 24, 1978, now U.S. Pat. No. 4,239,680.

This invention relates to peptides having biological activity and to peptides which can be converted to such biologically active substances. More particularly, the invention relates to substances having calcitonin-like biological activity or which may be converted to substances having such calcitonin-like activity. The invention deals also with processes for the preparation of these substances.

BACKGROUND

All known natural calcitonin peptides are similar in structure, and all contain an amino acid sequence of 32 amino acids. Salmon calcitonin, for example, has the following formula: ##STR1##

In our U.S. Pat. No. 3,926,938 we disclose the synthesis of the salmon calcitonin above referred to, and in our U.S. Pat. No. 3,929,758 we disclose improved processes by which peptides of this character may easily be prepared.

SUMMARY OF INVENTION

We have discovered a synthetic peptide with 31 amino acids that has calcitonin-like biological activity. Structurally, this peptide is similar to the natural calcitonins, particularly to salmon calcitonin. One significant difference in structure is that in our new peptides the amino acid sequence does not contain the tyrosine residue at position 22 of the salmon calcitonin amino acid sequence. Chemically, our new peptide is more stable because it does not contain the oxidatively labile tyrosine residue. The new peptide is also more economically produced since it contains one less amino acid. The new peptide has a biological activity similar in potency and quality to salmon calcitonin.

DESCRIPTION OF INVENTION

In general we use a solid phase type of synthesis and start with a resin called benzhydryl amine resin (BHA resin). This resin is derived from a cross-linked polystyrene bead resin manufactured by copolymerization of styrene and divinylbenzene. Resin of this type is known and its preparation is further demonstrated by Pietta et al (Pietta, P. S. and Marshall, G. R., Chem. Commun., 650 [1970]). This cross-linked polystyrene BHA resin is available from chemical supply houses. We use the designation ##STR2## to represent the BHA resin in which R is the polystyrene portion of the resin.

Resin Peptide Synthesis

In this synthesis the amino acids are added one at a time to the insoluble resin until the total peptide sequence has been built up on the resin. The functional groups of the amino acids are protected by blocking groups. The α-amino group of the amino acids is protected by a tertiary butyloxycarbonyl group or an equivalent thereof. This α-tertiary butyloxycarbonyl group we designate as BOC. The hydroxyl functions of serine and threonine are protected by a benzyl or benzyl derivative group such as 4-methoxybenzyl, 4-methylbenzyl, 3,4-dimethylbenzyl, 4-chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl, benzhydryl or an equivalent thereof. We use the term BZ to represent benzyl or benzyl derivative group.

The thiol function of cysteine may be protected by benzyl or benzyl protective groups described above and designated BZ or by an n-alkylthio group such as methylthio, ethylthio, n-propylthio, n-butylthio or equivalents thereof. We use the character R₇ to represent an n-alkylthio group or BZ and the character R₁ to represent BZ when R₇ is n-alkylthio and to represent n-alkylthio when R₇ is BZ. The guanidino function of arginine may be protected by a nitro group, a tosyl group or an equivalent thereof. We use the character T to represent either a nitro group or a tosyl group. The ε-amino function of lysine may be protected by a benzyloxycarbonyl group or a benzyloxycarbonyl derivative such as 2-chlorobenzyloxycarbonyl, 2-bromobenzyloxycarbonyl, 3,4-dimethylbenzyloxycarbonyl or equivalents thereof. We use the character V to represent benzyloxy carbonyl group or a benzyloxycarbonyl derivative group. The protective groups used on the imidazole nitrogen of histidine are the benzyloxycarbonyl group and benzyloxycarbonyl derivatives such as described above for lysine and are designated as V. The γ-carboxylic acid group of glutamic acid is protected by a benzyl or benzyl derivative group such as described for the protection of the hydroxyl function of serine and threonine. Thse protective groups are represented by the character BZ.

The formula of our synthetic peptide having calcitonin-like activity may be written as follows: ##STR3##

As may be seen from the formula above given, 31 amino acids are involved and in this formula the positions are numbered according to the accepted custom beginning at position 1 for the CYS on one end of the chain and ending with PRO at position 31 at the other end of the chain. For clarity of description this same numbering system will be followed in referring to the cycles of the synthesis. The assembly of the amino acids begins with cycle 31 which involves the coupling of proline and continues with cycle 30 which involves the coupling of threonine, etc.

Preferred amino acid reactants for use in each of the 31 cycles of the synthesis are given in the following Table 1:

                  TABLE I                                                          ______________________________________                                         Cycle                                                                          Number  Amino Acid Reactant                                                    ______________________________________                                         31      BOC--L--proline                                                        30      BOC--O--benzyl-L--threonine                                            29      BOC--glycine                                                           28      BOC--O--benzyl-L--serine                                               27      BOC--glycine                                                           26      BOC--O--benzyl-L--threonine                                            25      BOC--L--asparagine p-nitrophenyl ester                                 24      BOC--O--benzyl-L--threonine                                            23      BOC--Ω-nitro-L--arginine or                                                BOC-Ω-tosyl-L--arginine                                        22      BOC--L--proline                                                        21      BOC--O--benzyl-L--threonine                                            20      BOC--L--glutamine p-nitrophenyl ester                                  19      BOC--L--leucine                                                        18      BOC--ε-CBZ-- L--lysine or BOC--ε-2-                              chlorobenzyloxycarbonyl-L--lysine                                    17      BOC--N(im)-CBZ-- L--histidine                                          16      BOC--L--leucine                                                        15      BOC--L--glutamic acid γ-benzyl ester                             14      BOC--L--glutamine p-nitrophenyl ester                                  13      BOC--O--benzyl-L--serine                                               12      BOC--L--leucine                                                        11      BOC--ε-CBZ-- L--lysine or BOC--ε-2-                              chlorobenzyloxycarbonyl-L--lysine                                    10      BOC--glycine                                                            9      BOC--L--leucine                                                         8      BOC--L--valine                                                          7      BOC--S--ethylthio-L--cysteine, BOC--S--                                          methylthio-L--cysteine, BOC--S--n-                                             propylthio-L--cysteine or BOC--S--                                             n-butylthio-L--cysteine                                               6      BOC--O--benzyl-L--threonine                                             5      BOC--O--benzyl-L--serine                                                4      BOC--L--leucine                                                         3      BOC--L--asparagine p-nitrophenyl ester                                  2      BOC--O--benzyl-L--serine                                                1      BOC--S--p-methoxybenzyl-L--cysteine, BOC--S--                                    benzyl-L --cysteine or BOC--S--3,4-                                            dimethylbenzyl-L--cysteine                                           ______________________________________                                    

Each of the amino acid derivatives mentioned in Table I may be purchased from supply houses except perhaps the derivative mentioned for use in cycle No. 7. These materials useful in cycle 7 may be prepared according to the method described in the literature (U. Weber and P. Hartter, Hoppe-Seyler's, Z. Physiol. Chem. 351, 1384-8 [1970]).

CYCLE 31

Coupling of Proline to BHA Resin

The reaction vessel used in all steps of the resin peptide synthesis may be a glass vessel equipped with inlet ports at the top for addition of materials and a sintered glass disk at the bottom for removal of soluble reaction mixtures and wash solvents by filtration. Filtration can be performed either by vacuum or the use of nitrogen pressure. The contents of the vessel can be agitated by shaking the entire vessel or by a mechanical stirrer.

In cycle 31 the BHA resin is placed in the reaction vessel and suspended in a solvent such as methylene chloride, chloroform, dimethylformamide, benzene or equivalents thereof in proportions of 3 to 12 ml of solvent per gram of resin. To this is added BOC-L-proline in an amount of 1 to 6 equivalents per free amine equivalent of the BHA resin employed. After a period of mixing of 5 to 10 minutes, a coupling reagent (CA) such as dicyclohexyl carbodiimide (DCC) is added. Other diimide coupling agents may be used. The diimide coupling agent is used in the amount of 0.5 to 2.0 equivalents per equivalent of BOC-L-proline used.

The BOC-L-proline may be coupled in the absence of a coupling reagent if its active ester derivative, its azide derivative, its symetrical anhydride derivative, or a suitably chosen mixed anhydride derivative is used. The active ester derivatives that may be employed are 2-nitrophenyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, N-hydroxysuccimide ester or equivalents thereof. The active esters are used in amounts of 1 to 10 equivalents per free amine equivalent of BHA resin.

The reaction mixture consisting of the BHA resin, the solvent, the BOC-L-proline, and the coupling reagent or BOC-L-proline active ester is stirred or shaken mechanically until the reaction is complete as is indicated by a ninhydrin test (E. Kaiser, et al., Anal. Biochem., 34 595-8 [1970]) on a test sample. After completion of the coupling reaction, the BOC-L-proline resin may be washed with solvents such a methylene chloride, chloroform, methyl alcohol, benzene, dimethylformamide, or acetic acid. The amount of wash solvent may suitably be 5 to 20 ml of solvent for each gram of BHA resin used initially. If it is desired to terminate the coupling reaction before completion, the washing procedure is used and the remaining free amino groups on the BOC-L-proline resin may be blocked from further reaction by acetylation with an excess of acetylation reagents. The acetylation procedure is performed by agitating the BOC-L-proline resin with a solution of the acetylation reagent for a period of 0.5 to 12 hours. Acetylation reagents such as N-acetylimidazole in methylene chloride solution or a mixture of acetic anhydride and triethylamine in chloroform can be used. The acetylation reagent may be used in the amount of 0.5 to 5.0 equivalents per equivalent of free amine titer of the starting BHA resin.

The coupling reaction to produce the BOC-L-proline resin may be described by the following formula: ##STR4##

Deprotection of BOC-L-proline Resin

The BOC-L-proline resin produced as above described may be washed with a solvent such as referred to above and deprotected by agitating it with an agent such as a mixture of trifluoroacetic acid (TFA) in a solvent such as methylene chloride, chloroform, benzene or equivalents thereof. The amount of TFA in the solvent can vary from 10 to 100% of the mixture. The amount of TFA-solvent mixture may vary from 3 to 20 ml per gram of BHA resin used initially. The reaction time may be from about 10 minutes to 4 hours. The deprotection step is terminated by filtration to remove the TFA-solvent mixture. The residual TFA may be removed from the L-proline resin by washing with 3 to 20 ml per gram of BHA resin of a solution of 5 to 30% of triethylamine in a solvent such as methylene chloride, chloroform, benzene or equivalents thereof. Other tertiary or secondary organic amines may be used in place of the triethylamine such as trimethylamine, N-ethylpiperidine, diisopropylamine or equivalents thereof. The free amine titer of the L-proline resin may be determined by the Dorman titration procedure (Dorman, L. C., Tetrahedron Letters, 1969 2319-21). The deprotection reaction may be described by the following formula: ##STR5##

CYCLE 30

The propyl BHA resin obtained as a result of cycle 31 may be suspended in a coupling solvent, the BOC-O-BZ-L-threonine derivative added and the mixture equilibrated in the same manner. The coupling agent, DCC, may be added, and after completion of the reaction as indicated by the ninhydrin test, the reaction mixture is removed from the BOC-O-BZ-threonylpropyl BHA resin by filtration. The peptide resin may be washed with solvents. The amounts of reactants and solvents and reaction times may be the same as described in cycle 31. The BOC group may be removed from the peptide resin by the deprotection method described in the cycle 31. The resulting O-BZ-threonylpropyl BHA resin is then ready for cycle 29. The reactions of the cycle 30 may be shown by the following formula: ##STR6##

For convenience, we may write this resulting resin peptide using abbreviated nomenclature as follows: ##STR7##

CYCLE 29

In cycle 29, the coupling reaction and also the deprotection reaction may be performed in the same manner as in cycle 30 except that BOC-glycine is used in place of BOC-O-BZ-L-threonine. The reaction through coupling and deprotection may be written: ##STR8##

CYCLE 28

In cycle 28, the coupling and deprotection reactions may be performed in the same manner as in cycle 30 except for the substitution of BOC-O-BZ-L-serine as the amino acid derivative. This may be written: ##STR9##

CYCLE 27

In cycle 27, the coupling and deprotection reactions are performed as described in cycle 30 except that BOC-glycine is substituted as the amino acid reactant. These reactions through coupling and deprotection may be written as follows: ##STR10##

CYCLE 26

In this cycle, the coupling and deprotection reactions may be as in cycle 30 using the same amino acid reactant, resulting in the following compound: ##STR11##

CYCLE 25

In cycle 25, the coupling reaction is performed using an active ester derivative of BOC-L-asparagine. The active ester procedure is used in place of the DCC coupling method to avoid a known side reaction that occurs with the use of DCC coupling agent with BOC-asparagine or BOC-glutamine. The reaction is performed using the active ester derivative of BOC-L-asparagine in the amount of 2 to 10 equivalents per free amine equivalent of BHA resin in dimethylformamide, mixtures of dimethylformamide with benzene, methylene chloride or chloroform or with equivalents thereof in amounts of 2 to 20 ml. of solvent per gram of BHA resin used initially. Reaction times are from 1 to 72 hours. The reaction mixture is removed from the BOC-peptide resin by filtration after completion of the reaction as indicated by a ninhydrin test. The active esters derivative employed may be 2-nitrophenyl esters, 4-nitrophenyl esters, pentafluorophenyl, or equivalents thereof. We use AE to designate the active ester portion of the derivative. The coupling reaction may be written: ##STR12## The deprotection reaction to remove the BOC group is performed as in cycle 31.

CYCLES 24-21

In each of cycles 24 to 21, the coupling and deprotection reactions may be conducted using the methods and proportions of reactants as in cycle 30 using BOC-BZ-L-threonine in cycle 24, BOC-ω-T-L-arginine in cycle 23, BOC-L-proline in cycle 22, and BOC-O-BZ-L-threonine in cycle 21. The compound resulting from the completion of cycle 21 may be written: ##STR13## This compound, to our knowledge, has never been reported and as will later be shown, may be converted to a peptide with calcitonin-like activity.

CYCLE 20

In cycle 20, the coupling and deprotection reactions may be performed using the methods and proportions of reactants as in cycle 25 using a BOC-L-glutamine active ester derivative as the amino acid derivative, resulting in the compound: ##STR14##

CYCLE 19

In cycle 19, the reactions are performed as in cycle 31 using BOC-L-leucine as the amino acid derivative. The compound resulting from cycle 19 is: ##STR15##

CYCLE 18

In cycle 18, we may use as the amino acid derivative BOC-ε-V-L-lysine. Otherwise, cycle 18 methods may be performed as in cycle 30 resulting in the compound: ##STR16##

CYCLES 17-15

Cycles 17 to 15 may be performed as in cycle 31 except for the use of BOC-N(im)-V-L-histidine in cycle 17, BOC-L-leucine as the reactant in cycle 16 and BOC-L-glutamic acid BZ ester (BZ represents the same groups as it represents for serine and threonine) as the reactant in cycle 15, resulting in the following compound from cycle 15: ##STR17##

CYCLES 14-8

Cycle 14 may be performed identically to cycle 20 using BOC-L-glutamine-AE as the amino acid derivative. Cycles 13 to 8 may be performed as in cycle 31 except for the use of BOC-O-BZ-L-serine in cycle 13, BOC-L-leucine in cycle 12, BOC-ε-V-L-lysine in cycle 11, BOC-glycine in cycle 10, BOC-L-leucine in cycle 9, and BOC-L-valine in cycle 8 resulting in the compound: ##STR18##

CYCLE 7

Cycle 7 may be performed as in cycle 31 except for the use of BOC-S-R-L-cysteine or for the amino acid derivative. The compounds resulting from cycle 7 are described by the formula: ##STR19## where R₇ is n-alkylthio.

CYCLES 6-2

Cycles 6 to 4 were performed as in cycle 31 except that BOC-O-BZ-L-threonine was used as the amino acid derivative in cycle 6, BOC-BZ-L-serine was used as the amino acid derivative in cycle 5 and BOC-L-leucine was used in cycle 4 as the amino acid derivative. Cycle 3 may be performed identically to cycle 26 using BOC-L-asparagine active ester. In cycle 2, the procedures may be the same as cycle using BOC-O-BZ-L-serine as the amino acid derivative. The compound resulting from cycle 2 is: ##STR20##

CYCLE 1

This cycle may be performed identically to cycle 7 using BOC-S-R-L-cysteine derivatives. The R group chosen for the cysteine may be the same as used in cycle 7 or different. For example, if the derivative chosen for cycle 7 is BOC-S-ethylthio-L-cysteine, the derivative in cycle 1 may be BOC-S-4-methoxybenzyl-L-cysteine or if BOC-S-4-methoxybenzyl-L-cysteine was chosen for cycle 7, then this derivative may be used also in cycle 1. The compounds resulting from cycle 1 are described by the formula: ##STR21## where one of R₁ and R₇ is s-n-alkyl and the other is BZ.

Cycle 1 represents the completion of the resin peptide. The resin peptide may be removed from the reaction vessel and dried in a vacuum. The weight of the resin peptide may be expected to be from 2.0 to 3.5 times weight of BHA resin used initially in the synthesis.

Resin Peptide Cleavage

The peptide is cleaved from the resin peptide resulting from cycle 1 by treatment with liquid hydrogen fluoride (HF). The HF cleavage reaction may be performed by treating a mixture of the resin peptide and anisole (0.5 to 5 ml for each gram of resin peptide) with liquid HF (2 to 20 ml for each gram of resin peptide) for 0.5 to 20 hours at -20° to +15° C. After the reaction period, the excess HF may be removed by evaporation and the resulting mixture of peptide and resin beads may be extracted with an organic solvent such as ethyl acetate, diethyl ether, benzene or the like to remove the anisole and residual of HF. The peptide may be separated from the resin beads by extraction into aqueous acetic acid. The peptide at this stage is not cyclic but is the non-cyclic product without the cyclic disulfide bond between the cysteines at positions 1 and 7 in the molecule.

The HF treatment removes all blocking groups from the peptide, except the S-alkylthio blocking groups on the thiol function of cysteine residue at position 7. The S-n-alkylthio-L-cysteine residue is stable to the HF cleavage procedure and remains intact throughout the cleavage and extraction procedures. The S-BZ-L-cysteine residue is cleaved by HF to yield a cysteine residue with a free thiol function. Both types of blocking groups have been employed during our synthesis in combination with each other at positions 7 and 1. Thus, the peptides obtained after HF cleavage can be one of two types depending upon the blocking groups chosen for the thiol function of the cysteine derivative used during the resin peptide synthesis.

If BOC-S-BZ-L-cysteine derivatives were used in the resin peptide synthesis cycle 1 and BOC-S-ethylthio-L-cysteine was used in Cycle 7, the peptide resulting after HF cleavage would be of Type 1 and would have a free thiol function at position 1 and have a S-ethylthio function on the cysteine residue at position 7. The peptide would be represented by the formula:

Type I ##STR22##

Conversely, if BOC-S-n-alkylthio-L-cysteine derivative was used in cycle 1 and the BOC-S-BZ-L-cysteines were used in position 7, the peptide resulting from the cleavage would be of the Type II and would be represented by the formula:

Type II ##STR23##

The conversion of Type I and II peptides to the cyclic disulfide peptide may be performed by diluting with distilled water the aqueous acetic acid solution of the crude peptides from HF cleavage to a final volume of 50 to 200 ml per gram of resin peptide cleaved. The pH of this solution is adjusted to 5 to 10 by the addition of ammonium hydroxide solution and the mixture is stirred in a closed container under a stream of an inert gas such as nitrogen for 2 to 48 hours. The reaction period can be stopped when the off-gas stream no longer contains n-alkylmercaptan. The pH of the reaction mixture may be lowered to 3.5 to 5.5 by the addition of glacial acetic acid.

The peptide has now been converted to a peptide with calcitonin-like biological properties. The crude peptide solution obtained can be purified chromatographically to yield a freeze-dried product similar in chemical properties and biological activity to natural salmon calcitonin.

Purification of the Crude Synthetic Calcitonin

The crude peptide solutions at pH 5.0 from the above synthesis may be concentrated using an ion-exchange procedure. The concentrate may be purified by a combination of gel-filtration procedures and ion-exchange chromatography methods. The final purified product may be obtained from solution by freeze-drying as a fluffy white solid. The product gives the correct amino acid analysis for the desired peptide.

Following is the specific example of the preparation of the peptide.

EXAMPLE 1

Resin Activation

The BHA resin (5 g) with an amine titer of 0.61 meq/g was placed in the reactor vessel of a peptide synthesizer marketed by Schwarz-Mann, Inc. of Orangeburg, New York. The resin was treated with 25 ml of the following solvents filtering after each treatment:

Methylene chloride for 2 minutes

Chloroform for 2 minutes two times each

10% triethylamine in chloroform for 5 minutes two times each

Chloroform for 2 minutes

Methylene chloride for 2 minutes three times each

Cycle 31

Coupling: The BHA resin, 25 ml. of methylene chloride and 1.31 g. (0.0061 moles) of BOC-L-proline was stirred for 10 minutes. 6.1 ml. of a methylene chloride solution of dicyclohexylcarbodiimide (1 milliequivalent of DCCI per 1 ml. of solution) was added to the reactor and the mixture agitated for 6 hours. The reaction mixture was removed from the reactor by filtration and the BOC-prolyl BHA resin subjected to the following successive 2 minute, 25 ml. washes, removing the wash by filtration each time;

Methylene chloride two times

Methyl alcohol two times

Methylene chloride three times

Acetylation: The resin was then agitated with a mixture of 1.5 ml. of triethylamine (TEA), 1 ml. of acetic anhydride and 25 ml. of chloroform for 2 hours. The reaction mixture was removed by filtration and the resin subjected to the following 2 minute, 25 ml. washes:

chloroform two times

Methyl alcohol two times

Methylene chloride three times

Deprotection: The BOC-protected resin was agitated for 5 minutes with a mixture of 15 ml. of trifluoroacetic acid (TFA) and 15 ml. of methylene chloride. This mixture was removed by filtration and the resin was agitated with a second mixture of 15 ml. of TFA and 15 ml. of methylene chloride for 30 minutes. The reaction mixture was removed by filtration and the resin subjected to the following 25 ml. washes:

Methylene chloride two times two minutes each

Methyl alcohol two times two minutes each

Chloroform two times two minutes each

10% TEA in chloroform two times ten minutes each

Chloroform two times two minutes each

Methylene chloride two times two minutes each

The L-proline BHA resin was titrated to establish the amine or proline titer. This value was 0.55 milliequivalents of amine or proline per gram of resin.

Cycle 30

Coupling: The L-prolyl resin, 25 ml of methylene chloride and 1.64 g (0.0053 mole) of BOC-O-benzyl-L-threonine were agitated for 10 minutes. Then 5.5 ml of a methylene chloride solution of dicyclohexylcarbodiimide (1 milliequivalent of DCCI per 1 ml of solution or a total of 0.0055 mole of DCCI) was added to the reactor and the mixture agitated for 2 hours. The reaction mixture was removed from the reactor and the resin was subjected to the following successive 2 minute, 25 ml washes, removing the wash by filtration each time.

Methylene chloride two times

Methyl alcohol two times

Methylene chloride three times.

A ninhydrin test was negative.

Deprotection: The deprotection procedure described in Cycle 31 was repeated for this cycle.

Cycles 29 through 26

The coupling and deprotection procedures used in these cycles were the same as in Cycle 30 except that the following amino acid derivatives were used in place of the threonine derivative:

Cycle 29--0.93 g (0.0053 mole) of BOC glycine

Cycle 28--1.55 g (0.0053 mole) of BOC-O-Benzyl-L-serine

Cycle 27--The reactant used was the same as Cycle 29

Cycle 26--The reactant used was the same as Cycle 30.

Cycle 25

Coupling: The peptide resin obtained from Cycle 26 was washed twice with 25 ml portions of dimethylformamide (DMF). The resin was then agitated for 24 hours with a solution of 2.82 g (0.008 mole) of BOC-L-asparagine p-nitrophenyl ester in 35 ml of DMF. The reaction mixture was filtered and the resin peptide subjected to two minutes washes with two successive 25 ml portions of the following solvents: DMF, methylene chloride, methanol, methylene chloride. Each individual solvent was removed by filtration. A ninhydrin test was negative.

Deprotection: The deprotection procedure used in Cycle 31 was repeated.

Cycle 24

Coupling and deprotection procedures were the same as in Cycle 30 using the same reactants and amounts.

Cycle 23

Coupling: The resin peptide obtained from Cycle 25 was washed with two successive 25 ml. portions of DMF. The resin peptide was then agitated for 10 minutes with a mixture of 3.42 g (0.008 mole) of BOC-N-γ-tosyl-L-arginine and 25 ml of DMF. Then 8 ml of DCCI in methylene chloride (equivalent to 0.008 mole of DCCI) was added and the mixture agitated for 6 hours. The reaction mixture was removed by filtration. The resin peptide was subjected to two minute washes with two successive 25 ml portions of the following solvents: DMF, methylene chloride methyl alcohol, methylene chloride. The ninhydrin test was negative.

Deprotection: Repeat deprotection procedures used in Cycle 31.

Cycle 22

Coupling: The peptide resin obtained from Cycle 23 was agitated for 10 minutes with 1.72 g (0.008 mole) of BOC-L-proline and 25 ml of methylene chloride. 8 ml. of DCCI in methylene chloride (equivalent to 0.008 mole of DCCI) was added and the mixture agitated for 6 hours. The reaction mixture was removed by filtration and the resin peptide subjected to two minute washes with two successive 25 ml portions of the following solvents: methylene chloride, methyl alcohol, methylene chloride. Each individual wash was removed by filtration. The ninhydrin test was negative.

Deprotection: The deprotection procedure used in Cycle 31 was repeated.

Cycle 21

The coupling and deprotection procedures used in these cycles were the same as in Cycle 22 except that in the coupling reaction the following amino acid derivative was used in place of BOC-L-proline.

Cycle 21--2.47 g (0.008 mole) of BOC-O-benzyl-L-threonine

Cycle 20

This procedure is the same as Cycle 25 except that 2.94 g (0.008 mole) of BOC-L-glutamine p-nitrophenyl ester is used in place of the asparagine derivative.

Cycles 19 through 15

The procedure is the same as used in Cycle 30 except that the following amino acid derivatives were used in place of the threonine derivative:

Cycle 19--1.32 g (0.0053 mole) of BOC-L-leucine

Cycle 18--2.20 g (0.0053 mole) of BOC-ε-2-chlorocarbobenzyloxy-L-lysine

Cycle 17--2.06 g (0.0053 mole) of BOC-N(im)-carbobenzyloxy-L-histidine

Cycle 16--See Cycle 19

Cycle 15--1.79 g (0.0053 mole) of BOC-L-glutamic acidγ-benzyl ester

Cycle 14

Same as Cycle 20.

Cycle 13

That procedure used was the same as used in Cycle 22 except that in the coupling reaction 2.36 g (0.008 mole) of BOC-O-benzyl-L-serine was used in place of the proline derivative.

Cycles 12 through 9

The procedures used were the same as used in Cycle 30 except in the coupling reactions the following amino acid derivatives were used in place of the threonine derivative.

Cycle 12--Same reactants as used in Cycle 19

Cycle 11--The reactants were the same as in Cycle 18

Cycle 10--Same reactants as used in Cycle 29

Cycle 9--Same reactants as used in Cycle 19

Cycle 8

Coupling: The resin peptide from Cycle 9 was agitated for 10 minutes with 1.74 g (0.008 mole) of BOC-L-valine and 25 ml of methylene chloride. Then 8 ml of DCCI in methylene chloride (equivalent to 0.008 mole of DCCI) was added and the mixture agitated for 16 hours. The reaction mixture was removed by filtration. The resin peptide was subjected to two minute washes with two successive 25 ml portions of the following solvents: methylene chloride, methyl alcohol, methylene chloride. Each individual wash was removed by filtration.

Deprotection: See Cycle 31.

Cycle 7

The procedure was the same as used in Cycle 30 except that in the coupling reaction 1.59 g (0.0053) of BOC-S-ethylthio-L-cysteine was used in place of the threonine derivative.

Cycle 6

The reactants and procedures used were the same as Cycle 30.

Cycle 5

The reactants and procedures used were the same as Cycle 28.

Cycle 4

The reactants and procedures used were the same as Cycle 19.

Cycle 3

The reactants and procedures used were the same as Cycle 25.

Cycle 2

The reactants and procedures used were the same as Cycle 28.

Cycle 1

The procedures used were the same as used in Cycle 30 except that 1.81 g (0.0053 mole) of BOC-S-p-methoxybenzyl-L-cysteine was used in place of the threonine derivative.

After completion of Cycle 1, the resin peptide was washed with two successive 25 ml. portions of n-hexane. The peptide material was removed from the reactor and dried in an electric vacuum oven at 40° C. and 0.1 mm of Hg for 24 hours.

Cleavage with Hydrogen Fluoride

The dried resin peptide (10 g) and 10 ml of anisole were placed in a Teflon reaction vessel. The vessel equipped with a Teflon-coated magnetic stirrer was placed in a dry ice-acetone bath and 75 ml. of hydrogen fluoride gas was condensed into the vessel. This mixture was stirred at 0° C. in an ice bath for 1 hour. The hydrogen fluoride was removed by evaporation at reduced pressure. The residue was triturated with six 75 ml portions of ethyl acetate. The peptide was extracted from the resin beads with 600 ml of 0.1 molar aqueous acetic solution.

Cyclization of the Peptide

The aqueous acetic acid extract obtained from hydrogen fluoride cleavage was diluted to 1.2 liters by addition of 70 ml of distilled water. The pH of the solution was adjusted to 7.5 by the addition of concentrated ammonium hydroxide. The solution was stirred in a closed vessel under a stream of nitrogen for 24 hours. At this time no ethyl mercaptan could be detected in the emerging nitrogen stream. The ethyl mercaptain content of the nitrogen stream was measured by passing the stream through a solution of Ellman's (Ellman, G. L., Arch. Biochem. Biophys., 82, 70-7 (1959). The pH of the reaction mixture was adjusted to 5.0 by addition of glacial acetic acid.

Purification of the Crude Hentriacontapeptide

The 1.2 liters of solution from the above synthesis at pH 5.0 was concentrated using a SP-Sephadex C-25 ion-exchange column. The 75 ml concentrate removed from the column with 0.5 molar sodium chloride solution was desalted and purified by passing through a Sephadex G-25 (fine) gel-filtration column and eluting with 0.03 molar aqueous acetic acid solution. The hentriacontapeptide fraction from this column was adjusted to pH 6.0 by addition of ammonium hydroxide solution. This solution was further purified by ion-exchange chromatography using a Whatman CM52 column eluted with ammonium acetate buffer. The hentriacontapeptide fraction from this column was adjusted to pH 5.0 by addition of glacial acetic acid. This solution was concentrated using a SP-Sephadex C-25 ion-exchange column. The 30 ml concentrate removed from the column with 0.5 molar sodium chloride solution was desalted with Sephadex G-25 (fine) gel-filtration column. The purified peptide fraction was collected and freeze-dried. The product was obtained as a fluffy white solid. Amino acid analysis of the product gave the following ratios with the theoretical values given in parenthesis: Asp 1.9 (2), Thr 5.4 (5), Ser. 4.1 (4), Glu 2.9 (3), Pro 2.1 (2), Gly 3.0 (3), Val 0.8 (1), Leu 5.0 (5), His 0.9 (1), Lys 1.9 (2), Arg 0.9 (1). This product assayed at 6000 MRC units per mg.

While only certain embodiments of our invention have been described in specific detail it will be apparent to those skilled in the art that many other specific embodiments may be practiced and many changes made be made, all within the spirit of the invention and the scope of the appended claims. 

We claim:
 1. A resin peptide having the following structure: ##STR24## in which Bz is benzyl, 4-methoxybenzyl, 3,4-dimethylbenzyl, 4 chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl or benzhydryl,T is nitro or tosyl, and R is divinylbenzene crosslinked polystyrene.
 2. A resin peptide having the following structure: ##STR25## in which Bz is benzyl, 4-methoxybenzyl, 3,4-dimethylbenzyl, 4 chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl or benzyhydryl,T is nitro or tosyl, V is benzyloxycarbonyl, 2-bromobenzyloxycarbonyl or 2-chlorobenzyloxycarbonyl, one of R₁ and R₇ is S-n-alkyl and the other is BZ, and R is divinylbenzene crosslinked polystyrene.
 3. A resin peptide having the following structure: ##STR26## in which BZ is benzyl, 4-methoxybenzyl, 3,4-dimehtylbenzyl, 4-chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl or benzylhydryl,T is nitro or tosyl, V is benzyloxycarbonyl, 2-bromobenzyloxycarbonyl or 2-chlorobenzyloxycarbonyl, R₇ is Bz or S-n-alkyl, and R is divinylbenzenes crosslinked polystyrene. 